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Conjugate for the specific targeting of anticancer agents to tumor cells or tumor vasculature and production thereof

a technology of tumor vasculature and anticancer agent, which is applied in the direction of growth factors/regulators, pharmaceutical non-active ingredients, animal/human proteins, etc., can solve the problems of compromising the benefits of treatment, affecting the survival rate of patients, and the failure of existing therapies for such internal cancers

Inactive Publication Date: 2009-12-10
THE BOARD OF RGT UNIV OF OKLAHOMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the therapies now available for internal cancers often give rise to side effects so harmful that they compromise the benefits of treatment, and existing therapies for such internal cancers often fail in many cases.
Radiation and surgery are limited in that they cannot treat widespread metastases that eventually form full-fledged tumors at numerous sites.
Unfortunately, the most common cancers (breast, lung, colorectal, and prostate cancer) are not yet curable with chemotherapy alone.
The problem with this approach is that normal cells with the receptors bound by the fusion protein are also killed, resulting in potentially severe side effects.
However, the current methioninase experimental methodologies require large dosages of methioninase as well as methionine-, homocystine-, and choline-restricted diets.
There is currently no method for targeting anticancer agents such as methioninase specifically to the surface of cancer cells, or specifically to the surface of blood vessels supplying the cancer cells.

Method used

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  • Conjugate for the specific targeting of anticancer agents to tumor cells or tumor vasculature and production thereof
  • Conjugate for the specific targeting of anticancer agents to tumor cells or tumor vasculature and production thereof
  • Conjugate for the specific targeting of anticancer agents to tumor cells or tumor vasculature and production thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0104]Expression and Purification of ATF-methioninase. A pKK223-3 plasmid containing the gene for L-methioninase (containing 398 amino acids and with a calculated molecular weight of 42.7 kDa) from Pseudomonas putida was kindly provided by Dr. Dennis Carson of the University of California, San Diego (Hori et al., 1996). Plasmid pULB1221 containing the gene for human urokinase was kindly provided by Dr. Paul Jacobs of the Free University of Brussels, Belgium (Jacobs et al., 1985). Plasmid pKK223-3, with the tac promoter and an ampicillin resistance gene, was obtained from Amersham Biosciences (Piscataway, N.J.). E. coli JM105 was used as the host for both vector construction and protein expression.

[0105]The following fusion protein gene was constructed:

N-(amino acids 1-49 of urokinase A chain)-Gly-Ser-Gly-Ser-Gly-Ser-(L-methioninase)-C

The amino acid sequence of the fusion protein was assigned SEQ ID NO:1, while the nucleic acid sequence of the fusion protein was assigned SEQ ID NO:2....

example 2

[0130]The work described in Example 1 demonstrates that ATF-methioninase fusion protein specifically binds to the urokinase receptor on MCF-7 breast cancer cells in vitro, based on the measurement of ATF-methioninase displaced by urokinase at various concentrations from the surface of the MCF-7 cells. ATF-methioninase produced a dose-dependent inhibition of both the proliferation and migration of MCF-7 cells in vitro over a period of 1 to 3 days.

[0131]Because of these results with ATF-methioninase, an expanded study was performed that compares the effect of ATF-methioninase on MCF-7 in vitro cell proliferation and migration to L-methioninase without the receptor targeting peptide added, and to ATF-methioninase with the methioninase mutated so that there is no enzymatic activity. The effect of ATF-methioninase on the cell proliferation and migration of PC-3 prostate cancer and SK-LU-1 lung cancer cells was also studied. To compare the effect of ATF-methioninase with a fusion protein ...

example 3

Binding of Recombinant L-Methioninase-Annexin V to Phosphatidyl Serine Immobilized on Plastic or on the Surface of Cancer Cells

[0166]In this example, the binding of annexin V, part of the fusion protein L-methioninase-annexin V, to phosphatidylserine (PS) by two different methods was demonstrated. In the first method, PS was immobilized on plastic microtiter plates. In the second method, PS was exposed on the surface of human breast cancer cells by the addition of hydrogen peroxide. Binding was measured using an antibody to L-methioninase. The proteins used in this study were produced by recombinant DNA technology in Escherichia Coli bacteria and were purified to homogeneity.

Procedures for Preparation of Proteins

[0167]The protein genes were cloned into E. coli on the vector pET-30 Ek / LIC, which incorporates a His6 tag at the N-terminus and an HRV 3C protease site just before the start of the desired protein. L-methioninase and annexin V are connected by the flexible linker Gly-Ser-G...

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Abstract

A conjugate is disclosed herein, wherein the conjugate includes a ligand having the ability to specifically and stably bind to an external receptor or binding site on a tumor vasculature endothelial cell, wherein the external receptor or binding site is specific for tumor vasculature endothelial cells. The conjugate also includes an anticancer agent that is selectively toxic to cancer cells operatively attached to the ligand. The anticancer agent may be L-methioninase. Pharmaceutical compositions comprising the conjugate are also disclosed, as well as methods of treating a cancer tumor or cancer cells with a therapeutically effective amount of the conjugate.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. Ser. No. 11 / 712,140, filed Feb. 28, 2007; which claims benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 60 / 777,725, filed Feb. 28, 2006. This application is also a CIP of U.S. Ser. No. 10 / 870,832, filed Jun. 17, 2004; which claims benefit of U.S. Provisional Application No. 60 / 479,106, filed Jun. 17, 2003. The contents of each of the above-referenced patent applications are hereby expressly incorporated in their entirety herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]Not applicable.BACKGROUND OF THE INVENTION[0003]Although the rate of cancer incidence has declined since 1990, the number of people in the U.S. who are expected to die in 2004 from cancer is still expected to exceed half a million. The five most prevalent types of cancer in the U.S., ranked by the estimated number of new cases for the year 2004 (excluding base and squamous c...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/51A61P35/00C12N5/06
CPCA61K38/51A61K47/48246A61K47/48269C07K14/485C07K14/49C07K2319/75C07K14/5406C07K14/5412C07K14/65C07K14/78C07K2319/55C07K14/52A61K47/64A61K47/642A61P35/00
Inventor HARRISON, ROGER G.PENTO, J. THOMASLIND, STUART E.
Owner THE BOARD OF RGT UNIV OF OKLAHOMA
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